JP2009145326A - Carrier for use in measurement of analyte, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To allow a carrier for measuring an analyte to quickly and easily detect the analyte with high sensitivity. <P>SOLUTION: A first reactant 4 having a reaction site 2 specifically combined with the analyte and a fluorescently labeled site 3, and a second reactant 7 having a reaction site 5 specifically combined with the analyte and a recognition site 6 for recognizing fluorescence emitted from the fluorescently labeled site 3 of the first reactant 4 are immobilized on the carrier in such a position relationship that each of the reactants can bind to the analyte. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a test substance measuring carrier using fluorescence resonance energy transfer and a method for producing the same.

Conventionally, as a method for detecting a protein, an immunoassay method using an antibody against the protein has been widely used. For example, the ELISA method can quantitate the target protein with high sensitivity, and the Western blot method detects a specific protein from a protein mixture by combining the excellent resolution of electrophoresis and the high specificity of an antigen-antibody reaction. be able to. In addition, by allowing a labeled antibody to act on a tissue specimen, the localization of the target protein in the tissue or cell can be observed.
However, these immunochemical methods require complicated operations such as antibody binding and subsequent washing.

  On the other hand, methods using various fluorescent proteins typified by green fluorescent protein (GFP) are known as methods for observing protein dynamics in vivo. Since these fluorescent proteins can be expressed in a living body by genetic manipulation, they are useful for observing the localization and dynamics of endogenous proteins in tissues and cells.

  However, in a method using such a fluorescent protein, the target protein must be expressed in a form fused with the fluorescent protein in advance in a living cell, and the foreign antigen in the cell cannot be visualized.

  Various studies have been made to solve these problems. For example, in biological systems, fluorescence resonance energy transfer or the like is often used to measure the spatial proximity of labeled biomolecules or molecular groups. This method involves various biological reactions or interactions of interest, such as protein-protein interactions, antigen-antibody reactions during immune reactions, receptor-ligand interactions, nucleic acid hybridization (hybridism). ), Or as a proof of measure for protein binding to nucleic acids.

  Patent Document 1 describes a method for detecting in real time by a competitive reaction with a foreign antigen using an immunoassay reagent in which an antigen and an antibody are connected by a linker and a fluorescent substance is labeled on each. In this immunoassay reagent, when there is no competing antigen, fluorescence resonance energy transfer (FRET) occurs due to the formation of the antigen-antibody complex, but when there is a competing antigen, the competing antigen binds to the antibody. Therefore, since FRET does not occur, it is described that the presence or absence of such competing antigens can be detected quickly and easily.

  However, in Patent Document 1, since the antigen-antibody complex is not fixed with a carrier, the washing operation cannot be performed, and there is a problem that the immunoassay reagent gradually diffuses and flows out in a non-sealed space. It can only be used once, and it was difficult to use it for repeated use. In addition, since it is a detection by the competitive method, the sensitivity tends to be poor, and it is insufficient for detection of a trace amount of antigen. In the competitive method in which another reagent is added later and measured, for example, measurement in the body Not suitable for. In addition, when two reactants are connected via a linker as in Patent Document 1, the linker must be designed each time so that the reactant can act effectively, and the versatility of the antigen is lost. There is a problem of becoming higher. For repeated use, this linker can be removed and separated and simply immobilized on a carrier, the distance between the first luminescent substance YFP and the second luminescent substance CFP can be made closer. However, if the fixed amount is increased in order to make them close to each other, there is a problem that a large amount of YFP and CFP are close to each other and the S / N ratio is extremely deteriorated.

  On the other hand, as a method for immobilizing an antigen on a solid layer using an antigen-antibody reaction, for example, Patent Document 2 discloses a technique called molecular imprinting. More specifically, a complex of a monomer or polymer in which a ligand for the target molecule is introduced and the target molecule is reacted with a polymer cross-linking agent to prepare a gel containing the complex of the ligand and the target molecule, It is described that a molecular imprint gel can be obtained by removing a target molecule from this gel. Further, it has been disclosed that when AFP as an antigen is added to a gel carrier containing an anti-AFP antibody and a lectin, the gel shrinks due to the binding of the anti-AFP antibody and the lectin to AFP. Describes that AFP (target molecule) can be detected.

However, since the antigen-antibody complex is immobilized on the carrier by polymerization, in order for the antigen to reach the antibody, it must enter the cross-linked gel matrix structure. For this reason, it takes time for the antigen to react with the antibody, and a very high concentration of antigen must be contacted for 2 to 4 hours in order to reliably recognize the substance. Furthermore, when antigens are washed and removed, for the same reason, they cannot be removed efficiently, and in addition, responsiveness tends to deteriorate. Therefore, even if the fluorescence resonance energy transfer method or the like is applied to the present technology, the distance change between the fluorescent labeling site and the recognition site that recognizes the fluorescence emitted from the fluorescent labeling site is very slow. There is a problem that the change in the fluorescence intensity cannot be measured at all times, and cannot be detected quickly and with high sensitivity.
JP 2007-40834 A JP 2006-138656 A

As described above, any of the above-described techniques is insufficient as a test substance-measuring carrier that can measure the target molecule in a short time with high sensitivity and simplicity and can be used repeatedly after washing.
In addition, when used as a biosensor, a highly versatile production method has been desired from the viewpoint of production cost.

The present invention has been made in view of such circumstances, and without using a competitive reaction, for example, using fluorescence resonance energy transfer or chemical amplification luminescence on a solid layer, a test object can be quickly and highly enhanced. An object of the present invention is to provide a test substance-measuring carrier that can be detected easily and sensitively and can be used repeatedly after washing.
Another object of the present invention is to provide a production method capable of producing such a test substance measuring carrier with high versatility and efficiently without designing a linker.

  The test substance measurement carrier of the present invention comprises a reaction site that specifically binds to a test substance and a fluorescent labeling site, a reaction site that specifically binds to the test object, and the first reactant. A second reactant having a recognition site for recognizing fluorescence emitted from a fluorescent labeling site is fixed on a carrier in a positional relationship capable of binding to the specimen.

  It is preferable that a polymer layer is provided on the carrier, and the first reactant and the second reactant are fixed on the carrier via the polymer layer. The thickness of the polymer layer is preferably 1 nm or more and 0.5 mm or less.

The polymer layer is preferably fixed on the carrier via a self-assembled film. The thickness of the self-assembled film is preferably 0.2 nm or more and 10 μm or less.
The polymer layer is preferably a polysaccharide.

The first reactant and the second reactant are preferably an antibody or a test substance-binding fragment. The fluorescent labeling site of the first reactant is preferably a fluorescent dye or a fluorescent protein. The reaction between the fluorescent labeling site of the first reactant and the recognition site of the second reactant is preferably fluorescence resonance energy transfer. The test object is preferably an antigen.
The test substance measurement carrier is suitable as a carrier used in a biosensor or a bioreactor.

  The method for producing a test substance-measuring carrier of the present invention includes a test substance, a reaction site specifically binding to the test substance and a first reactant having a fluorescent labeling site, and a reaction specifically binding to the test substance. And a second reactant having a recognition site for recognizing fluorescence emitted from the site and the fluorescence labeling site of the first reactant, to obtain a conjugate of the first reactant, the test substance and the second reactant. The bonded substance is applied to the surface of the carrier, and the first reactant and the second reactant are immobilized on the surface of the carrier, respectively.

It is preferable that after removing the first reactant and the second reactant on the surface of the carrier, the test object is removed from the bound product.
The removal of the test object is preferably performed under conditions that reduce binding of the test object.

  The carrier for measuring a test object of the present invention includes a first reactant having a reaction site and a fluorescence labeling site that specifically binds to the test object, and a fluorescence label of the reaction site and the first reactant that specifically binds to the test object. Since the second reactant having a recognition site for recognizing fluorescence emitted from the site is fixed on the carrier in such a positional relationship that it can bind to the test sample, it flows out of the carrier even if a washing operation is performed. There is no end.

  In addition, each of the first reactant and the second reactant has a reaction site that specifically binds to the test substance, and is independently fixed on the carrier in a positional relationship that can bind to the test article. Therefore, when the specimen is present, it can be easily combined in a short time. Since the recognition efficiency of fluorescence is changed when the first and second reactants recognize the test sample, the concentration of the test sample can be measured accurately and simply by measuring this amount of change. It becomes. In addition, since the first reactant and the second reactant are immobilized on the carrier, they do not diffuse or flow out even if a washing operation is performed, and repeated measurement is possible.

  Furthermore, since it is not the detection by a competitive method, it is possible to measure a very small amount of a test substance with high sensitivity. Furthermore, the first reactant and the second reactant need only have a reaction site that specifically binds to the test object, respectively, and it is not necessary to design a linker for each test object as in Patent Document 1, Further, in the fluorescent substance labeling, since it is not necessary to selectively label the fluorescent substance and the fluorescent recognition substance on the same molecule, the present invention does not need to connect the first reactant and the second reactant with a linker, Therefore, since it can be applied to any antigen-antibody combination and labeling can be performed separately, there is an advantage that it is very versatile and excellent in manufacturing suitability.

  Hereinafter, the test substance measuring carrier of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram showing an embodiment of the test substance-measuring carrier of the present invention. As shown in FIG. 1, the test substance measurement carrier of the present invention comprises a reaction site 2 that specifically binds to the test substance and a first reactant 4 having a fluorescent labeling site 3 and a reaction that specifically binds to the test substance. The second reactant 7 having the recognition site 6 for recognizing the fluorescence emitted from the site 5 and the fluorescence labeling site 3 of the first reactant 4 has a positional relationship on the carrier 11 so as to be able to bind to one specimen. The first reactant 4 and the second reactant 7 are fixed to the hydrogel 13 through the silane coupling layer 12 and the hydrogel 13 provided in FIG.

  FIG. 1 shows a mode in which the bonded product 10 is fixed to the hydrogel 13 via the silane coupling layer 12 and the hydrogel 13 provided on the carrier 11, but depending on the material of the carrier 11, The coupling layer 12 and the hydrogel 13 may not be provided, or only one of them may be provided.

  The fluorescence labeling site 3 of the first reactant 4 and the recognition site 6 that recognizes the fluorescence emitted from the fluorescence labeling site 3 of the first reactant 4 of the second reactant 7 cause fluorescence resonance energy transfer between them. It is a part. Fluorescence resonance energy transfer is a phenomenon in which excitation energy moves between two fluorescent dyes having different excitation wavelengths (a fluorescent labeling site 3 possessed by the first reactant 4 and a fluorescence recognition site 6 possessed by the second reactant 7). Since the first reactant 4 and the second reactant 7 are fixed to the carrier 11 so as to be capable of binding to the test object, the first and second reactants 4 and 2 are labeled with each other. When the test substance binds to the fluorescence recognition site 6 of the reactant 7, the distance becomes close to each other and fluorescence resonance energy transfer occurs.

  This will be described with reference to FIG. FIG. 2 is a schematic diagram showing a state in which the specimen is combined in FIG. In FIG. 2, the same components as those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted unless necessary (hereinafter the same). When the test substance is added to the test substance measurement carrier shown in FIG. 1, the reaction site 2 that specifically binds to the test substance 1 of the first reactant 4 and the test substance 1 and the peculiarity of the second reactant 7 Reaction sites 5 that bind to each other specifically bind to the specimen 1, whereby the fluorescent labeling site 3 of the first reactant 4 and the fluorescence recognition site 6 of the second reactant 7 are close to each other. Here, fluorescence resonance energy transfer occurs. As a result, the fluorescence of the fluorescent labeling site 3 of the first reactant 4 decreases and the fluorescence of the fluorescence recognition site 6 of the second reactant 7 increases. Therefore, by observing the change in the fluorescence intensity before and after the addition of the specimen 1, the specimen 1 can be detected (measured).

  Here, the case where the reaction between the fluorescent labeling site 3 of the first reactant and the recognition site 6 of the second reactant is fluorescence resonance energy transfer has been described as an example. The reaction between the fluorescent labeling site 3 and the recognition site 6 of the second reactant is not limited to this. For example, the first reactant excites oxygen molecules, and the second reactant is excited by the excited singlet oxygen. Chemically amplified luminescence or the like that emits light at the recognition site may be used.

  Next, a method for producing the test article measurement carrier of the present invention will be described with reference to FIG. The test substance measurement carrier of the present invention forms a conjugate of the first reactant, the test subject, and the second reactant, and then the conjugate is placed on the carrier on which the hydrogel is immobilized by the method described below. Obtained by applying and immobilizing and removing the test substance from the conjugate.

  Since the first reactant 4 and the second reactant 7 have reaction sites 2 and 5 that specifically bind to the test object 1, the conjugate of the first reactant 4, the test object 1, and the second reactant 7 10 can be obtained by mixing the first reactant 4, the test substance 1 and the second reactant 7 in an arbitrary ratio in an aqueous solution.

  Next, the bound substance 10 can be immobilized on the carrier surface by adding the bound substance 10 to the surface of the carrier 11 and reacting it appropriately. At this time, since the reaction sites 2 and 5 of the first reactant 4 and the second reactant 7 are protected by the binding of the test object 1, it is not necessary to perform a special protection process separately. The method of binding this conjugate to the carrier will be apparent to those skilled in the art. For example, when a carboxyl group is present on the surface of the carrier, a method of activating and immobilizing the surface of the carrier with an activator such as EDC described later, a maleimide group is imparted to the surface of the carrier, and the first reactant and / or second Although the method of making the thiol group which a reactant has react and immobilize can be taken, in the present invention, it is not limited to these methods and arbitrary methods can be taken.

When used for a biosensor, the fixed amount (density) of the bound substance bound on the test substance-measuring carrier is preferably 1 piece / mm ³ to 1 × 10 ¹⁸ pieces / mm ³ . More preferably, it is 1 × 10 ⁹ pieces / mm ³ to 1 × 10 ¹⁵ pieces / mm ³ , and further preferably 1 × 10 ¹⁰ pieces / mm ³ to 1 × 10 ¹⁴ pieces / mm ³ . When this density is actually measured, the bound substance is immobilized on the support, the test object is washed, and the fluorescence amounts of the first reactant fluorescent label and the second reactant fluorescent label are determined. Each is obtained by measuring and calculating the film thickness by AFM or ellipsometry.

  In order to obtain the test substance-measuring carrier of the present invention shown in FIG. 1, the test substance 1 may be removed from the bond 10 after immobilizing the bond 10 on the carrier 11, and preferably the first reaction. The reaction site 2 of the body 4 and the test substance 1 and the reaction site 5 of the second reactant 7 and the test substance 1 are reduced in binding to the carrier of the first reactant 4 and the second reactant 7. It is performed under conditions that do not reduce binding. In detail, what is necessary is just to wash | clean with respect to the support for test article measurement. At this time, since the first reactant 4 and the second reactant 7 are independently immobilized on the carrier, the subject 1 can be easily removed, and the subject when used as an immobilized carrier. The bond reproducibility with 1 is not reduced.

  The removal of the test object can be easily performed by using an appropriate washing solution. The washing solution used here may be any solution that reduces the binding force of the test object. Such conditions for reducing the binding force can include changing the pH to the acidic or alkaline side, increasing the salt concentration, and the like. The conditions vary depending on the type of the first reactant, the second reactant, the test substance, etc. For example, an acidic glycine buffer, an alkaline NaOH solution for adjusting the pH to 2 or less or 10 or more, 0.5 M or more Examples thereof include a borate buffer for obtaining a salt concentration. In addition, an arginic acid-containing acidic buffer, a guanidine-containing buffer, or the like can be used as appropriate. Here, the washing treatment with the washing liquid can be appropriately adjusted, but from the viewpoint of not impairing the binding activity between the first reactant and the second reactant, it is generally 10 minutes or less, preferably 1 minute or less. Preferably, it is more preferably 5 seconds or more from the viewpoint of reproducibility.

  Hereinafter, each component such as a carrier constituting the test object measurement carrier in the embodiment shown in FIG. 1 and the test article will be described, and then the case of applying to a biosensor and a bioreactor will be described.

(1) Carrier The carrier of the present invention is generally an optical glass such as BK7, a metal oxide such as silica, alumina, titania, zirconia, or indium tin oxide (ITO), silicon nitride, gallium nitride, or aluminum nitride. , Metal nitrides such as indium nitride, metal oxides such as indium tin oxide (ITO), or synthetic resins, specifically sepharose, polyethylene, polystyrene, poly (meth) acrylic acid, poly (meth) acrylamide, polymethyl It is desirable that (meth) acrylate, polyethylene terephthalate, polycarbonate, or cycloolefin polymer is provided with a functional group. In particular, those made of a material transparent to a light source such as glass, ITO, polymethyl (meth) acrylate, polyethylene terephthalate, polycarbonate, and cycloolefin polymer are more preferable. Examples of functional groups include amino groups, carboxyl groups, maleimide groups, aldehyde groups, succinimide groups, thiol groups, hydrazine groups, isocyanate groups, epoxy groups, vinyl sulfone groups, vinyl groups, and cyan groups. .

Such a carrier is more preferably a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.
As a method for imparting these functional groups, a known surface treatment method using a plasma treatment, an ozone treatment, an acid / alkali etching treatment, a self-assembled film, or the like can be adopted. It is more preferable to use a self-assembled film.

(2) Polymer layer A polymer layer is bonded onto the carrier. The polymer layer can be composed of a hydrophilic polymer, a hydrophobic polymer, or a combination thereof, but it is preferable to use only a hydrophilic polymer. The polymer layer may be bonded directly on the carrier or indirectly. In the case of direct bonding, for example, there is a method of bonding a polymer from a carrier via graft polymerization. As an indirect bonding method, a hydrophobic polymer is applied on a carrier and then a hydrophilic polymer is bonded. The polymer layer may be bonded after a carrier and a compound capable of binding to the polymer (hereinafter referred to as a linker) are applied to the surface of the carrier. According to a particularly preferable embodiment, a self-assembled film can be used as a linker, and a hydrophilic polymer can be bonded to the glass carrier via the self-assembled film. Hereinafter, this aspect will be described.

(2-1) Self-assembled film Examples of the self-assembled film include (1) a method using a silane coupling agent and (2) a method using alkanethiol. Each method will be described below.

(1) Method of using a silane coupling agent For a substrate having a metal oxide or nitride such as silica or silicon nitride or a thin film thereof, through a silane coupling layer formed by the silane coupling agent It can also be combined. As a silane coupling agent, general formula A-1 (in general formula A-1, X ^a represents a functional group, La represents a linker moiety containing ^a linear, branched, or cyclic carbon chain, and R ^a represents Hydrogen or an alkyl group having 1 to 6 carbon atoms, Y ^a represents a hydrolyzable group, and m and n each represents an integer of 0 to 3, and m + n = 3). By utilizing this, the substrate surface can be coated with a functional group by forming a covalent bond such as carrier (silicon) -oxygen-silicon-carbon on the carrier surface.

Here, examples of the hydrolyzable group (Y ^a ) include an alkoxy group, a halogen, and an acyloxy group, and more specifically, a methoxy group, an ethoxy group, chlorine, and the like. A methoxy group and an ethoxy group are preferable. Specific examples of the silane coupling agent include γ-aminopropyltrimethoxysilane, N-β (aminoethyl) γ-aminopropyltrimethoxysilane, γ-aminopropylmethyldiethoxysilane, γ-mercaptopropyltrimethoxysilane, Examples thereof include γ-glycidoxypropyltriethoxysilane. As a reaction method of the silane coupling agent, a general method may be used. For example, the method described in Books, Effects and Usage of Silane Coupling Agent (Science & Technology) can be used.

Further, the functional group (X ^a ) possessed by the silane coupling agent is not particularly limited as long as it binds to a polymer or a bond described later, and is an amino group, a carboxyl group, a hydroxyl group, an aldehyde group, a thiol group, an isocyanate. Groups, isothiocyanate groups, epoxy groups, cyano groups, hydrazino groups, hydrazide groups, vinyl sulfone groups, vinyl groups, maleimide groups, and combinations and derivatives thereof can be used. (X ^a ) is an amino group and an epoxy group.

(2) Method of using alkanethiol In the method of using alkanethiol, a metal film is arranged on the above-described carrier, and then alkanethiol is imparted. Here, the term “arranged on the carrier” means that the metal film is arranged via another layer without being in direct contact with the carrier, in addition to the case where the metal film is arranged so as to be in direct contact with the carrier. It also means including the case. Examples of the metal constituting the metal film include free electron metals such as gold, silver, copper, aluminum, and platinum, and gold is particularly preferable. These metals can be used alone or in combination. In consideration of adhesion to the carrier, an intervening layer made of chromium or the like may be provided between the carrier and the layer made of metal.

  The thickness of the metal film is arbitrary, but is preferably 0.1 nm or more and 500 nm or less, and particularly preferably 1 nm or more and 200 nm or less. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 0.1 nm or more and 10 nm or less.

The coating method of metal film using alkanethiol has been developed vigorously by Professor Whitesides et al. Of Harvard University, and its details are reported in, for example, Chemical Review, 105, 1103-1169 (2005). When gold is used as the metal, an alkanethiol represented by general formula A-2 (in general formula A-2, n represents an integer of 3 to 20 and X represents a functional group) is used as the organic layer forming compound. Thus, based on the van der Waals force between the Au—S bond and the alkyl group, a monomolecular film having orientation is formed in a self-organized manner. The self-assembled film is produced by a very simple technique in which a gold carrier is immersed in a solution of an alkanethiol derivative. Specifically, for example, in the general formula A-2, ^Xb is an amino group, carboxyl group, hydroxyl group, aldehyde group, thiol group, isocyanate group, isothiocyanate group, epoxy group, cyano group, hydrazino group, hydrazide group, By forming a self-assembled film using a compound that is a vinyl sulfone group, a vinyl group, or a maleimide group, a functional group can be imparted to the surface of the carrier.

  In the above general formula A-2, the repeating number n of the alkyl group is more preferably an integer of 3 to 16, particularly preferably an integer of 4 to 8. Further, the alkyl group portion may be substituted with a multiple bond or a hetero element such as nitrogen or oxygen. When the alkyl group of the alkanethiol derivative is short, it is difficult to form a self-assembled film, and when the alkyl group is long, water solubility is lowered and handling becomes difficult.

In addition, the alkanethiol represented by the general formula A-2 can form a self-assembled film with only one type of functional group ^Xb , and can be mixed with a plurality of types of alkanethiols to form a self-assembled film. It is also possible to form.
By using such a linker, a carrier having ^a functional group (X ^a ) or a functional group (X ^b ) on the surface can be produced. Here, the linker does not have to be a self-assembled film as long as it is a compound capable of imparting a functional group (X ^a ) or a functional group (X ^b ) to the surface of the carrier.

  The thickness of such a self-assembled film is arbitrary, but is preferably 0.2 nm or more and 10 μm or less, particularly preferably 1 nm or more and 500 nm or less, and more preferably 100 nm or more and 500 nm or less. If the thickness is 10 μm or less, the test object can easily diffuse in the membrane, and if the thickness is 0.2 nm or more, the amount of the target substance to be fixed can be increased.

  In the present invention, the first reactant and the second reactant can be directly immobilized on the above-described self-assembled film. However, the viewpoint of improving the test substance binding rate of the first reactant and the second reactant. From the above, it is preferable to form a polymer layer on the self-assembled film and provide a functional group for fixing the first reactant and the second reactant on the surface of the carrier.

(2-2) Polymer As the polymer that can be used in the present invention, a hydrophilic polymer can be preferably used. Specifically, gelatin, agarose, chitosan, dextran, carrageenan, alginic acid, starch, cellulose, or these Or a water-swellable organic polymer such as polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol or derivatives thereof.

  As the hydrophilic polymer used in the present invention, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be further used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, for example, JP-A-59-53836, page 3, line 20 to page 6, line 49, JP-A-59-71048. Methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer, maleic acid copolymer, partial ester as described in the specification, page 3, line 41 to page 7, line 54 And maleic acid copolymerization, and those obtained by adding an acid anhydride to a polymer having a hydroxyl group. The carboxyl group-containing polysaccharide may be any one of an extract from a natural plant, a product of microbial fermentation, an enzymatic synthesis, or a chemical synthesis. Specifically, hyaluronic acid, chondroitin sulfate, heparin, Examples include dermatanic acid sulfuric acid, carboxymethylcellulose, carboxyethylcellulose, ceurouronic acid, carboxymethylchitin, carboxymethyldextran, and carboxymethyl starch. As the carboxyl group-containing polysaccharide, a commercially available compound can be used. Specifically, carboxymethyldextran, CMD, CMD-L, CMD-D40 (manufactured by Meisho Sangyo Co., Ltd.), sodium carboxymethylcellulose (sum) Kogaku Pharmaceutical Co., Ltd.), sodium alginate (Wako Pure Chemicals Co., Ltd.), and the like.

The weight average molecular weight of the hydrophilic polymer used in the present invention is not particularly limited, but generally it is preferably 2 × 10 ² or more and 5 × 10 ⁵ or less, more preferably 1 × 10 ⁴ or more and 2 × 10 ⁶ or less. Preferably there is. When the weight average molecular weight is smaller than this range, the fixed amount of the reactant may be small, and when the weight average molecular weight is larger than this range, handling may be difficult due to high solution viscosity.

  The hydrophilic polymer bonded to the sensor surface preferably has a film thickness in an aqueous solution of 1 nm to 0.5 mm, more preferably 1 nm to 1 μm, and still more preferably 100 nm to 500 nm. When the film thickness is thin, the amount of physiologically active substance immobilized decreases, and interaction with the analyte substance hardly occurs. On the other hand, if the film thickness is large, the uniformity of the hydrophilic polymer cannot be maintained, which is not preferable. The hydrophilic polymer film thickness in an aqueous solution can be evaluated by AFM, ellipsometry, or the like.

(2-3) Activation of polymer When using the polymer containing a carboxyl group as a polymer, it can couple | bond with the support | carrier surface by activating a carboxyl group. As a method of activating a polymer containing a carboxyl group, a known method, for example, a method of activating with water-soluble carbodiimide 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) Alternatively, a method of activation with EDC alone can be preferably used. By reacting a polymer containing a carboxyl group activated by this method with a carrier having an amino group, a hydrophilic polymer can be bound on the carrier.

Further, as a method for activating a polymer containing a carboxyl group, there is a method using a nitrogen-containing compound. Specifically, the following general formula (Ia) or (Ib) (wherein R ¹ and R ² are Independently represents a carbonyl group which may have a substituent, a carbon atom or a nitrogen atom, R ¹ and R ² may form a 5- to 6-membered ring by a bond, and A is a carbon atom having a substituent Alternatively, a nitrogen-containing compound represented by a phosphorus atom, M represents an (n-1) -valent element, and X represents a halogen atom can be used.

Here, R ¹ and R ² each independently represent a carbonyl group, a carbon atom, or a nitrogen atom which may have a substituent. Preferably, R ¹ and R ² have a 5- to 6-membered ring by a bond. Form. Particularly preferably, hydroxysuccinic acid, hydroxyphthalic acid, 1-hydroxybenzotriazole, 3,4-dihydroxy-3-hydroxy-4-oxo-1,2,3-benzotriazine, and derivatives thereof are provided.
Also preferably used are the nitrogen-containing compounds shown below.

Preferably, as the nitrogen-containing compound, a compound represented by the following general formula (I) (wherein Y and Z independently represent CH or a nitrogen atom) can also be used.

As specific compounds of general formula (I), the following compounds are preferred.

Moreover, as a nitrogen-containing compound, the following compounds etc. are mention | raise | lifted preferably.

Preferably, the nitrogen-containing compound is represented by the following general formula (II) (wherein A represents a carbon atom or phosphorus atom having a substituent, and Y and Z independently represent CH or a nitrogen atom) , M represents an (n-1) valent element, and X represents a halogen atom.

  Here, as the substituent of the carbon atom or phosphorus atom represented by A, an amino group having a substituent is preferable, and a dialkylamino group such as a dimethylamino group or a pyrrolidino group is preferable. Examples of the (n-1) -valent element represented by M include a phosphorus atom, a boron atom, and an arsenic atom, and a phosphorus atom is preferable. Examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom is preferable.

Preferred specific compounds of the general formula (II) include the following compounds.

The nitrogen-containing compound includes the following general formula (III) (wherein A represents a carbon atom or phosphorus atom having a substituent, M represents an (n-1) -valent element, and X represents a halogen atom). Can also be used.

As specific compounds of general formula (III), the following compounds are preferred.

Further, as a method for activating a polymer containing a carboxyl group, it is also preferable to use a phenol derivative having an electron withdrawing group, and the σ value of the electron withdrawing group is preferably 0.3 or more. Specifically, the following compounds can be used.

  These carbodiimide derivatives and nitrogen-containing compounds or phenol derivatives are not only used in combination, but can also be used alone if desired. Preferably, a carbodiimide derivative and a nitrogen-containing compound are used in combination.

Moreover, the following compound can also be used as a method of activating the polymer containing a carboxyl group. Although this compound can also be used independently, you may use together with a carbodiimide derivative, a nitrogen-containing compound, and a phenol derivative.

  Furthermore, as a method for activating carboxylic acid in a polymer containing a carboxyl group, the method described in JP-A-2006-58071, “0011” to “0022” (that is, specifying a carboxyl group present on the surface of a carrier) And a method of forming a carboxylic acid amide group by activation using any one of a uronium salt, a phosphonium salt, or a triazine derivative having the structure: and JP-A-2006-90781, “0011” to “0019” (Ie, a carboxyl group present on the surface of the carrier is activated with a carbodiimide derivative or a salt thereof, and has a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or a thiol group. After making an ester with any of the aromatic compounds, react with amines to form carboxylic acid amide groups That way) can be used preferably.

(2-4) Application of polymer to carrier In the present invention, when a polymer containing a carboxyl group is used as the polymer, the polymer containing an activated carboxyl group may be reacted with the carrier as a solution. Alternatively, the reaction may be performed in a state where a thin film on the carrier is formed using a technique such as spin coating. Preferably, the reaction is in a state where a thin film is formed.

  As described above, the polymer containing a carboxyl group activated in the present invention is preferably reacted with a carrier in a thin film state. As a method for forming a thin film on a carrier, known methods can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, and a spray coating method. , Slide bead coating method, slit and spin method, slit coating method, die coating method, dip coating method, knife coating method, blade coating method, flow coating method, roll coating method, wire bar coating method, transfer printing method, etc. It is possible. For these thin film formation methods, "Progress in coating technology" by Yuji Harasaki, Comprehensive Technology Center (1988), "Coating Technology" Technical Information Association (1999), "Aqueous Coating Technology" CMC (2001), "Evolution" Described in "Organic Thin Film Deposition", Sumibe Techno Research (2004), "Polymer Surface Processing" by Iwamori Satoshi, Gihodo Publishing (2005), etc. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a carrier in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

(3) First reactant and second reactant In the present invention, the first reactant and the second reactant have a reaction site that specifically binds to the test substance. It is a test substance binding fragment. The antibody or antigen-binding fragment thereof used in the present invention is a Fab fragment or F (ab ′) ₂ fragment having binding properties to the antibody molecule itself and the corresponding antigen, and each of the L chain and H chain of the antibody. Artificial single chain antibodies such as ScFV (single chain fragment of variable region) constructed by connecting variable regions are included. Single chain antibodies are preferred because they can be expressed in cells other than mammals such as prokaryotic cells such as Escherichia coli and plant cells, and because their volume is small, fluorescence resonance energy transfer is likely to occur. Further, the L chain and H chain of the antibody can be used as a combination of the first reactant or the second reactant, respectively. In addition, a method for producing a single chain antibody such as ScFV itself is well known. The antibody or antigen-binding fragment thereof used in the present invention is not particularly limited as long as it retains the binding property by the antigen-antibody reaction with the test substance (antigen) described later, and the antibody obtained by using the antigen as an immunogen or its antigen It is not limited to binding fragments. The antibody used in the present invention reacts with a test substance by antigen-antibody reaction.

  In the present invention, the recognition of fluorescence broadly includes being excited directly from the excited state of the fluorescent substance or indirectly excited to cause luminescence or chemical reaction. Both the first reactant and the second reactant in the present invention may be labeled with a fluorescent substance, or only the labeled antibody may be labeled with a fluorescent substance. When the first reactant and the second reactant are labeled with a fluorescent substance, they may be present at any site as long as they are labeled at a site that does not inhibit the binding with the antigen or the binding with the carrier. . The recognition site of the second reactant capable of recognizing the fluorescence of the fluorescent material is not limited to the fluorescent material and is not particularly limited as long as it can be used repeatedly.

  As described above, when the fluorescent labeling portion of the first reactant is irradiated with light having an excitation wavelength, the fluorescent labeling portion of the first reactant and the fluorescent recognition portion of the second reactant are close to each other. In this case, fluorescence resonance energy transfer occurs, the fluorescence of the fluorescent labeling site of the first reactant decreases, and the fluorescence of the fluorescence recognition site of the second reactant increases. Does not occur, the fluorescence of the fluorescent labeling site of the first reactant increases, and the fluorescence of the fluorescence recognition site of the second reactant decreases.

  Here, fluorescence includes light emission from molecules in an excited state generated by a chemical reaction, such as phosphorescence or luminol. For example, firefly (Photinus pyralis) luciferin, Cypridina luciferin, Renilla reniformis luciferin, luminescent earthworm (Diplocardia) luciferin, lathia (Latia neritoides) luciferin, firefly squid luciferin (Watfelin) In addition to luciferin derived from luminescent organisms such as), aequorin derived from Aequorea is known.

  The above-described fluorescence resonance energy transfer is well known as shown in JP-T-2007-523754 [0158] to [0161] and JP-T 2001-519525 [0025], and is a fluorescent dye for fluorescence resonance energy transfer. Various fluorescent labeling sites possessed by the first reactant and fluorescence recognition sites possessed by the second reactant are also commercially available. As the fluorescent labeling site and the fluorescent recognition site, any combination can be used as long as fluorescence resonance energy transfer occurs between them, and it can be freely selected from information such as literatures or commercially available products. For example, fluorescein derivative, rhodamine derivative, aminocoumarin derivative, hydroxycoumarin derivative, BODIPY derivative, anthracene derivative, benzofuran derivative, porphyrin derivative, Cy Dye, Alexa Fluor, europium cryptate, DyLight, HiLyte Fluor, Oyster, MegaStokes Dye, IRDye fluorescence Examples of dyes and fluorescent proteins include DFP, YFP, GFP, and BFP.

  When using fluorescence resonance energy transfer in the present invention, it is desirable that energy transfer occurs between the selected fluorescent substances and that the energy transferred state is stable. GFP-YFP, Alexa Fluor555-Alexa Fluor 647 A combination of fluorescent proteins or fluorescent dyes such as FITC-Alexa Fluor 555 can be preferably used. These combinations may be fluorescent proteins only, fluorescent dyes only, or a combination of fluorescent dyes and fluorescent proteins, and are not limited to these examples. From the viewpoint of stability of fluorescence emission, Alexa Fluor and HiLyte Flour are preferable, and Alexa Fluor is more preferable.

  In addition, it is widely known in fluorescence resonance energy transfer between CFP-YFP, between GFP-BFP, and between these variants, and these fluorescent proteins are also commercially available. If a fluorescent protein is used, it can be produced as a single molecule fusion protein by linking to a labeled antibody or the like (antibody or antigen-binding fragment thereof). When used in living cells, a gene encoding the fusion protein can be introduced. For this reason, fluorescent proteins are convenient when used in living cells. Nucleic acids encoding these fluorescent proteins are well known, and various vectors containing them are also commercially available. Therefore, fluorescently labeled proteins obtained by fusing a fluorescent protein to a desired polypeptide use those commercially available vectors. And can be easily prepared.

  The method of labeling an antibody or the like with a fluorescent dye is appropriately selected depending on the type of fluorescent dye used. When the fluorescent dye is a non-peptidic compound, it can be labeled by a known method such as chemical modification by attaching a reducing group such as maleimide to the thiol group or amino group of the antibody. When the fluorescent dye is a peptide compound such as a fluorescent protein, as described above, it can be produced as a fusion protein with an antibody or the like. The method for producing the fusion protein is also described in the following examples, but is not limited thereto, and can be produced using any known method.

  The fluorescent dyes possessed by the first reactant and the second reactant are bound to a position where fluorescence resonance energy transfer occurs when the antibody or the like and the test substance are bound by the antigen-antibody reaction. In order to bind a fluorescent dye to an antibody or the like, it may be bound directly or via a spacer. By using such a spacer, it is possible to appropriately adjust the position of the fluorescent dye so that fluorescence resonance energy transfer occurs when the antibody or the like and the test substance are bound by an antigen-antibody reaction.

(5) Test article The test article of the present invention is preferably an antigen in order to bind specifically to the reactant. The type of antigen is not particularly limited as long as it can interact with an antibody or the like, and can be appropriately selected according to the purpose as a detection substance. However, since it is necessary to bind to two reactants, it is necessary that there are two or more binding sites for the reactants.

(6) Application of the test substance measuring carrier of the present invention The test substance measuring carrier of the present invention is a biosensor bioreactor (for example, “Bioreactor Technology”, 1988, CMC Corporation, “Biochip and Biosensor ", 2006, Kyoritsu Shuppan Co., Ltd.). A biosensor is interpreted in the broadest sense, and means a sensor that measures and detects a target substance by converting an interaction between biomolecules into a signal such as an electrical signal. A normal biosensor is composed of a receptor site for recognizing a chemical substance to be detected and a transducer site for converting a physical change or chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity for each other is immobilized on a carrier and used as a molecular recognition substance, thereby selectively measuring the other substance to be matched.

  The biosensor to which the test substance measurement carrier of the present invention is applied cannot detect a test substance that is equal to or higher than the reactant constituting the binding substance immobilized on the carrier. In order to increase the dynamic range, it is preferable to measure the time course. By this time course measurement, the concentration in a wider region can be determined from the binding speed and the binding amount.

  In addition, since the test article measurement carrier of the present invention is a fixed carrier, even if the conjugate formed by a plurality of types of test articles is fixed, the plurality of types of test articles respectively correspond to the first reaction. A conjugate is formed between the body and the second reactant, and no conjugate is formed between the non-corresponding first and second reactants. Therefore, crosstalk, which is usually a problem in ELISA or the like, does not occur, and multiple items can be measured simultaneously.

A bioreactor is a reactor applied to production of useful substances, generation of energy, decomposition of environmental pollutants, and the like using biochemical reactions by biocatalysts such as enzymes, fungus bodies, cells, and organelles. In a bioreactor capable of producing a useful substance, reaction, etc. using an insoluble carrier having an enzyme immobilized thereon (for example, No. 4-18398, No. 4-18399, etc.) The sample measurement carrier of the present invention can be applied.
Examples of the test article measuring carrier of the present invention are shown below.

Example 1
<Preparation of carrier having amino group>
(Washing of carrier)
A slide glass manufactured by Matsunami Glass Industrial Co., Ltd. that had been irradiated with UV was immersed in acetone and ultrasonically cleaned for 5 minutes. Then, the slide glass was taken out from acetone and washed with pure water.

(Amino (APS) group attachment)
In a 100 ml glass vial, 72 ml of ethanol and 8 ml of pure water were mixed and heated at 60 ° C. for about 15 minutes in a thermostatic layer (manufactured by ASONE) to warm the mixed solution of ethanol and pure water. In a mixed solution of warm ethanol and pure water, 80 μl of APS (γ-aminopropyltriethoxysilane, manufactured by Tokyo Chemical Industry Co., Ltd.) was mixed (0.1% v / v) and dissolved. The washed slide glass was immersed in this solution and reacted at 60 ° C. for 15 minutes in a constant temperature layer (manufactured by ASONE) to obtain a slide glass provided with an APS group. The slide glass after completion of the reaction was taken out and washed by a step of immersing it in a solution of ethanol: pure water = 9: 1 and taking it out. This washing process was performed three times. The slide glass after washing was blown with nitrogen and baked at 120 ° C. for 1 hour. The slide glass after baking was immersed and washed in the order of ethanol and pure water.

<Active esterification of CMD>
After dissolving CMD (Carboxymethyldextran, manufactured by Meisei Sangyo: Molecular weight: 1 million) in ultrapure water, 2% of the carboxyl groups are activated when the total amount is reacted. M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide) and 0.1 M NHS (N-Hydroxysuccinimide) mixed solution were added and stirred at room temperature for 5 minutes.

<Production of CMD film>
An active esterified CMD solution was dropped onto the gold film on which the amino group had been formed and removed after 30 seconds, thereby forming an active esterified carboxymethyldextran thin film on the carrier having an amino group. After reacting at room temperature for 1 hour, it was washed once with 0.1 N NaOH and once with ultrapure water.

<Label to antibody>
1 mg Myoglobin primary antibody (10-M50c Fitzgerald Industries International) is labeled with Alexa Fluor (registered trademark) 555 (Molecular Probes) according to the manufacturer's recommended prescription to produce a primary labeled Myoglobin antibody. The antibody Myoglobin secondary antibody 1 mg (manufactured by 10-M50d Fitzgerald Industries International) was labeled with Alexa Fluor (registered trademark) 647 (manufactured by Molecular Probes) according to the manufacturer's recommended prescription to prepare a secondary labeled Myoglobin antibody.

<Immobilization of antibody>
The prepared primary-labeled Myoglobin antibody, secondary-labeled Myoglobin antibody and Myoglobin (10-M50 Fitzgerald Industries International) were mixed at a molar ratio of 1: 1: 1, and 0.1 with a pH 4.5 acetate buffer (Biacore). Prepared to mg / ml. 10 μl of the mixture was immobilized on the CMD membrane using a Biacore surface prep unit (μ channel), which is an option of Biacore 3000 (manufactured by GE Healthcare). At this time, the mixture was activated for 7 minutes using 70 μl of a mixed activation solution of 0.2 M EDC / 0.04 M NHS (manufactured by GE Healthcare). Thereafter, the plate was washed five times with PBS (phosphate buffered saline: manufactured by Invtrogen), 10 mM NaOH (manufactured by Wako Pure Chemical Industries), and pH 2.0 glycine buffer (manufactured by GE Healthcare).

  After that, the fluorescence of the CMD film was measured by FLA-8000 (manufactured by Fuji Film Co., Ltd.), and the intensity ratio between the light emission near 570 nm and the light emission near 675 nm was obtained. Thereto was added 0.1 mg / ml Myoglobin (in PBS buffer) for 3 minutes, and the mixture was flushed with pure water. Similarly, the fluorescence of the CMD film after flowing was measured with FLA-8000, and the intensity ratio between the light emission near 570 nm and the light emission near 675 nm was obtained. Divide the intensity ratio of the emission near 570 nm and the emission near 675 nm by the intensity ratio between the emission near 570 nm and the emission near 675 nm obtained before and after the addition of Myoglobin, and the change in the emission intensity ratio before and after the addition of Myoglobin The rate was determined. Table 1 shows the results.

(Example 2)
In Example 1, after fluorescence measurement, washing was repeated 5 times alternately with pH 2.0 glycine buffer and 10 mM NaOH. Thereafter, 0.1 mg / ml Myoglobin (in PBS buffer) was added in the same manner as in Example 1, and the change rate of the luminescence intensity ratio before and after the addition was determined.

(Comparative Example 1)
In Example 1 <Immobilization of antibody>, except that the mixed solution of primary labeled Myoglobin antibody, secondary labeled Myoglobin antibody and Myoglobin was fixed with only primary labeled Myoglobin antibody and secondary labeled Myoglobin antibody (no Myoglobin). Immobilization was carried out in the same manner as in Example 1, 0.1 mg / ml Myoglobin (in PBS buffer) was added in the same manner as in Example 1, and the change rate of the luminescence intensity ratio before and after the addition was determined.

  As is clear from Table 1, in Examples 1 and 2 in which primary and secondary labeled Myoglobin antibodies and Myoglobin were immobilized in advance with a mixed solution, the FRET intensity after addition of Myoglobin was changed, but a comparative example. No change in FRET intensity was observed in No. 1, and therefore, the test substance measurement carrier of the present invention (Examples 1 and 2) is composed of a primary labeled Myoglobin antibody, a secondary labeled Myoglobin antibody, and Myoglobin. In contrast, primary-labeled myoglobin antibody and secondary-labeled myoglobin antibody are close to each other at which fluorescence resonance energy transfer can occur, whereas in comparative example 1, primary-labeled myoglobin antibody and secondary-labeled myoglobin antibody It can be seen that the fluorescence resonance energy transfer is not fixed at a short distance. In addition, although an Example and a comparative example showed the luminescence intensity ratio change rate before and behind the addition of myoglobin of the same density | concentration and are numerically 3% of difference, in the comparative example, the density | concentration of myoglobin is 100 Even if it is doubled (10 mg / ml), it cannot be detected, and for the first time in the present invention, it has been demonstrated that the test substance can be detected by applying fluorescence resonance energy transfer to the solid phase.

Accordingly, as shown in the present invention, a conjugate composed of the first reactant, the test substance, and the second reactant is immobilized on the carrier with the first reactant and the second reactant. Since the test substance measurement carrier can apply fluorescence resonance energy transfer to a solid phase, rapid in situ measurement is possible.
Furthermore, it can be seen from Example 2 that the sample can be immobilized on the carrier even after washing the test article, and that repeated measurement is possible. Therefore, according to the test article measurement carrier of the present invention, a highly versatile immobilization carrier can be provided with high sensitivity and simplicity in a short time.

Schematic schematic diagram showing one embodiment of the test substance measurement carrier of the present invention Schematic schematic diagram showing the state in which the test article is removed in FIG. Process drawing which shows the manufacturing process of the test substance measurement support | carrier of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Test object 2 Reaction site 3 Fluorescence labeling site 4 1st reactant 5 Reaction site 6 Recognition site 7 2nd reactant
10 Combined
11 Carrier
12 Silane coupling layer
13 Hydrogel

Claims (14)

A first reactant having a reaction site that specifically binds to a test substance and a fluorescent labeling site;
A positional relationship in which a reaction site that specifically binds to the test object and a second reactant that has a recognition site that recognizes fluorescence emitted from the fluorescent labeling site of the first reactant can each bind to the test article. A carrier for measuring a test substance, which is fixed on the carrier.   The test object measurement according to claim 1, further comprising a polymer layer on the carrier, wherein the first reactant and the second reactant are fixed on the carrier via the polymer layer. Carrier.   3. The test substance measuring carrier according to claim 2, wherein the polymer layer has a thickness of 1 nm to 0.5 mm.   The test substance measurement carrier according to claim 2 or 3, wherein the polymer layer is fixed on the carrier through a self-assembled film.   5. The test substance measurement carrier according to claim 4, wherein the self-assembled film has a thickness of 0.2 nm to 10 μm.   The test substance measurement carrier according to claim 3, 4 or 5, wherein the polymer layer is a polysaccharide.   The test substance measurement carrier according to any one of claims 1 to 6, wherein the first reactant and the second reactant are an antibody or a test substance-binding fragment.   The test substance measurement carrier according to any one of claims 1 to 7, wherein the fluorescent labeling portion of the first reactant is a fluorescent dye or a fluorescent protein.   The test object according to any one of claims 1 to 8, wherein the reaction between the fluorescent labeling site of the first reactant and the recognition site of the second reactant is fluorescence resonance energy transfer. Carrier for measurement.   The test substance measurement carrier according to claim 1, wherein the test substance is an antigen.   The test substance measurement carrier according to any one of claims 1 to 10, wherein the test substance measurement carrier is a carrier used in a biosensor or a bioreactor. A test substance, a first reactant having a reaction site and a fluorescence labeling site that specifically bind to the test object, a reaction site that specifically binds to the test object, and a fluorescence labeling site of the first reactant. And reacting with a second reactant having a recognition site that recognizes fluorescence to obtain a conjugate of the first reactant, the test substance and the second reactant,
A method for producing a test substance-measuring carrier comprising applying the bound substance to a carrier surface and immobilizing the first reactant and the second reactant on the carrier surface, respectively.   13. The carrier for measuring a test substance according to claim 12, wherein the test substance is removed from the bound substance after the first reactant and the second reactant are immobilized on the surface of the carrier, respectively. Method.   The method for producing a test substance measurement carrier according to claim 12 or 13, wherein the removal of the test substance is performed under conditions that reduce binding of the test substance.